1. Field of the Invention
The present invention relates to high affinity and specific antibodies for a variant of human intact parathyroid hormone [hPTH (1-84)] (SEQ ID NO. 1) or its fragments which have at position 17 the amino acid serine which is phosphorylated [p(17) hPTH] and a method for preparing such antibodies. The invention further includes various immunoassay methods which use the described antibody alone or in combination with other antibodies to determine the circulating levels of intact p(17) hPTH (1-84) (SEQ ID NO. 1) or fragments thereof in biological fluids.
2. Background of the Invention
Parathyroid hormone (PTH) and its importance in regulating the concentration of calcium ions in the blood is well known. In this regard, PTH is created by the parathyroid glands and, in combination with other factors, functions to regulate the blood calcium ion levels such that they are maintained in a steady concentration in cells and the surrounding fluids. Essentially, PTH functions to release stored calcium in the body when serum calcium levels decrease. On the other hand, such secretion is suppressed to the extent serum calcium concentration increases.
In its complete bioactive form, hPTH comprises a unique peptide comprised of 84 amino acids (SEQ ID NO. 1). Given its significance in calcium metabolism, accurately measuring PTH has and continues to be of substantial clinical significance. As is well documented, serum PTH levels serve as an important parameter for patients having diseases such as hypercalcemia, primary hyperparathyroidism, and osteoporosis, among many others. PTH likewise becomes of substantial clinical importance in patients afflicted with chronic renal failure who, because of an excess in PTH production, can develop renal osteodystrophy.
Notwithstanding its important role in metabolism and clinical significance, substantial difficulties have and continue to exist with regard to determining circulating biologically active PTH levels. First of all, it is well known that PTH is normally present at extremely low levels, which are normally between 5 pg/mL to 45 pg/mL. Furthermore, it is well known that the PTH peptide is also present as a variety of circulating PTH fragments, including amino-terminally truncated fragments which are not bioactive. Second generation PTH assays were known to measure many of these non-bioactive fragments as well as the bioactive PTH, thereby leading to results that showed a PTH level higher than the actual circulating bioactive PTH levels. Third generation assays were able to overcome this shortcoming by binding to the amino-terminal end of PTH, thereby giving a more accurate reading of the bioactive PTH levels in a patient.
However, D'Amour et al. describe an amino-terminal form of hPTH that can be separated from hPTH (1-84) by HPLC. Amino-Terminal Form of Parathyroid Hormone (PTH) with Immunologic Similarities to hPTH (1-84) is Overproduced in Primary and Secondary Hyperparathyroidism, Clin Chem 49:12, 2037-2044 (2003). The article further postulates that this variant may be hPTH that is phosphorylated at the serine residue in position 17. An important finding of this paper is that the percentage of this new form of hPTH to intact hPTH (1-84) varied by disease state. Specifically, the new form of hPTH was lowest in the non-diseased control group, was increased in the renal failure patients, and was the highest in patients having primary hyperparathyroidism. Accordingly, there is a need in the art for an assay that can efficiently and cost-effectively measure this new form of hPTH.
Third generation bioactive hPTH assays that only bind to the first few amino acids of the amino terminal recognize this new form of hPTH along with intact hPTH (1-84). However, second generation hPTH assays that recognize both intact hPTH (1-84) and amino-terminally truncated hPTH fragments such as hPTH (7-84) have diminished recognition to this new form of hPTH. Since the antibodies utilized in second generation assays usually bind in the region of amino acid 17 of hPTH, this diminished recognition to hPTH is further suggestive of a possible modification, such as phosphorylation, at this amino acid site.
These binding characteristics could explain a phenomenon described in a recent article by Boudou et al. Unexpected Serum Parathyroid Hormone Profiles in Some Patients with Primary Hyperparathyroidism, Clin Chem 52:4, 757-760 (2006). Typically, second generation hPTH assays yield patient results that are higher than third generation hPTH results because the second generation assays detect amino-terminally truncated hPTH fragments in addition to intact hPTH (1-84). The article by Boudou et al. describes seven patients in which this ratio is reversed, that is the third generation assays produce a higher patient result than the second generation assays. This is potentially due to increased amounts of p(17) hPTH which was undetected by the second generation PTH assays. The seven patients presented clinically with severe primary hyperparathyroidism and the authors further speculate that this unexpected profile may be predictive of malignancy. As this study used only indirect evidence, an assay that can directly measure p(17) hPTH may prove extremely valuable for assessing hyperfunctional parathyroid gland activity.